1. Field of the Invention
The present invention is directed to the use of polyanilines, which undergo a color change upon oxidation or reduction, in the visual or spectrophotometric determination of the presence of oxidants or reductants in a sample.
2. Description of the Related Art
L-Ascorbic acid (vitamin C) occurs naturally in many foodstuffs (fruits, vegetables, dairy products, meat, etc.) and is frequently added to processed foods as an antioxidant. The production of ascorbic acid from fermentation is an important bioprocess which supplies most of the demands of the pharmaceutical as well as the food industry for ascorbic acid. Determination of ascorbic acid is important, due to both its nutritional value as a vitamin and oxygen scavenger, as a freshness indicator and as a control parameter during fermentation (see 1M. Olliver, The Vitamins 359-367(W. H. Sebrell & R. S. Harris eds., 2d ed. 1967)). Measurement of ascorbic acid levels in clinical samples (urine, blood, etc.) is also of interest, since ascorbic acid concentration is a useful indicator of several pathological states (see Nagy et al., 31 Life Sci. 2611-2616(1982); Schenk et al., 54 Anal. Chem. 1452-1454(1982); M. Brin, Ascorbic Acid: Chemistry, metabolism, and uses 369-379(P. A. Seib & B. M. Tolbert eds., 1982)).
Ascorbic acid is a moderately strong reducing agent in aqueous media (E.sub.1/2 =-0.104 V vs SCE at pH 5) (see Leinweber et al., 143 Methods Enzymol. 15-17(1987). Most analytical methods exploit its ease of oxidation, and deduce its concentration by spectrophotometric determination (see Farmer, 34 Proc. Soc. Exp. Biol. Med. 146-160(1936); Roe et al., 147 J. Biol. Chem. 399-407(1943); and Grote et al., 212 Anal. Chim. Acta 273-278 (1988)) or titration (see Tillmans, 54 Z. Unters. Lebensm. 33-38(1972); Leonhardt et al., 122 Z. Anal. Chem. 3-10(1941); and Sastry et al., 19 Talanta 212-220(1972)) of the redox products. For example, the dye 2,6 -dichlorophenolindophenol is often used as an oxidant in ascorbic acid determination, and its reduction is measured spectrophotometrically. An analytical problem occurs if other reductants of interest for foodstuffs, particularly sulfites, are also present in significant quantities in the ascorbic acid samples. In such cases the background noise from other reductants may constitute a large part of the total measurement. The indirect determination of ascorbic acid using biosensors based on immobilized ascorbate oxidase is the only reliable homogeneous assay (see Lloyd et al., 28Food Chem. 257-264(1988); Sidwell et al., 2 Biosensors 221-238(1986); and Carvallo et al., Process Biochem. 52-54(April, 1989)) besides the well established, but expensive and complicated HPLC method (see L-Ascorbinsaure, Boehringer Mannheim, Methoden der biochemischen Analytik und Lebensmittelanalytik 16-18(1987)). There are some application problems with the biological method as well. Firstly, ascorbic acid oxidase is an expensive enzyme (approximately $100/10 mg). Secondly, the inadequate stability of the enzyme, irrespective of the immobilization method, has always been a difficult problem in the development of biosensors. Lastly, the strongly reducing potential that is required for the operation of biosensors (approximately -0.94 V vs SCE) may lead to the co-reduction of background oxidants (see 49 M. L. Hitchman, Chemical Analysis (P. J. Elving & J. D. Winefordner eds., 1978)). Therefore, there has been a need to develop a simple, and yet highly selective method for the determination of ascorbic acid.